1. Field of the Invention
The present invention relates to a unique method for obtaining bacterium pili subunits suitable for use in preparing a vaccine.
2. Description of the Prior Art
Pili, also referred to as fimbriae, are hairlike organelles attached to the outer surface of bacteria. Pili are composed of many identical protein subunits arranged in a helical array. For pathogenic bacteria, pili are important for virulence, and their role appears to be that of promoting adherence of the bacterium to the host's tissues. That is to say, the importance of pili lies in the fact that they participate in the pathogenic process of many disease causing bacteria. The first step in any bacterial infection is attachment of the bacteria to the host's tissues. The second step is the colonization phase during which the bacteria divide and increase their numbers at or near the site of initial attachment. The third step is the actual pathogenic step which can vary from one bacterial species to the next. For example, in the case of enterotoxogenic E. coli, toxins are secreted by the bacteria which damage host tissue and cause fluids to move into the gut resulting in diarrhea.
As revealed in the prior art patents and professional publications, several vaccines have already been developed and are being developed utilizing pili as immunogens. Because pili are important in the early adhesion stages of infections, these vaccines are thought to act to prevent the subsequent colonization step and, thereby, to subvert the infection. Literally all known vaccines today contain intact pili.
The strains of bacteria used to prepare such vaccines are selected on the basis of pilus serotype. In the case of N. gonorrhoeae bacteria, the serotype of these pili is highly variable. Therefore, great care must be used in the selection of the strains selected to produce the vaccine to obtain the broadest range of protection. There is a low level of cross reactivity among the pili produced by the different strains, but compared to the antigenicity of the variable portions of the pili subunit, this is minor. Thus, although such an "intact pilus" vaccine gives good protection against infection with the homologous strain of bacteria, it gives much poorer protection against heterologous strains.
Exemplar prior art patent teachings concerned with vaccines utilizing a pili component are Brinton's U.S. Pat. No. 4,237,115 and U.S. Pat. No. 4,298,597 to Acres. Both these patents disclose vaccines of an "intact pilus" type. Othe analogous prior art disclosures are contained in the following U.S. Pat. Nos.:
Homma, U.S. Pat. No. 3,928,565 PA1 Fullerton, U.S. Pat. No. 4,199,565
Buchanan, U.S. Pat. No. 4,203,971
Karkhanis, U.S. Pat. No. 4,220,638
Helting, U.S. Pat. No. 4,271,147.
Just as a study of prior patent literature reveals pertinent disclosures, so also does a review of prior professional literature. For example, this inventor recognized that pili might be further degraded into subunits in his paper titled, "Isolation of and Some Physical-Chemical Properties of Pili Isolated from Type 2 Colonies of Neisseria gonorrhoeae," which appeared in PILI, D. E. Bradley, Ed., International Conferences on Pili, Washington, D.C. 1978. Further pertinent prior art disclosure has also been made by this inventor in his article titled, "Structure of Common Pili from Escherichia coli," JOURNAL OF BACTERIOLOGY, Vol. 138, No. 3, June 1979 at 969-975. In the second article, this inventor discusses not only a method for obtaining pili, but also means for disrupting purified pili into subunits, or pilin. Isaacson, et al, in their article titled, "Escherichia coli K99 Pili Are Composed of One Subunit Species," FEMS MICROBIOLOGY LETTERS, 12 at 229-232 (1981), confirm the work of this inventor as reported by him in Volume 138 of the JOURNAL OF BACTERIOLOGY, and present further findings concerning the structure of K99 subunits. More recently, Korhonen and Rhen, in their article titled, "Bacterial Fimbriae as Vaccines," ANNALS OF CLINICAL RESEARCH, Vol. 14 at 272-277 (1982), have recognized the limitations of "intact pilus" vaccines and have recognized that a subunit vaccine would probably exhibit greater efficacy against heterologous strains.
Thus, it is clear that vaccines prepared from intact, or whole, pili are well known in the prior art. It is also accurate to state that the prior art has recognized the hypothetical desirability of preparing pili subunit vaccine. It is postulated that in such a subunit vaccine, the antigenic zones (epitopes) which are identical for the pili from one strain to the next would be exposed. Accordingly, such a subunit vaccine would permit a greater degree of cross protection against different bacterial strains.
The primary difficulties associated with producing a pili subunit vaccine have basically involved determining a commercially-acceptable procedure for deriving the pili subunits so that a subunit vaccine can be readily prepared in accord with accepted manufacturing processes.